The present invention relates to a decomposition product of bacterial cell walls, an immunomodulator, consisting of bacterial cells or their decomposition materials which can suppress selectively IgE antibody production, and to provide their production methods and foods containing them.
Immediate type allergies or atopies, such as pollen allergy, asthma, atopic dermatitis, food allergy, are known to be mediated by antibody of IgE isotype immunoglobulin. Symptoms of atopy are elicited when the IgE antibodies react to antigen, resulting in release of various physiologically active substances such as histamine, serotonin, leucotrienes and others.
For the prevention and treatment of immediate type allergies, drugs generally used today are those which are antagonistic to the mediators, for instance, antihistaminics, drugs which inhibit release of mediates from mast cells, and corticosteroids which inhibit activation of various immunocytes resulting in inhibition of cytokine production.
Since the antibody which mediates an immediate allergy is an IgE antibody, an agent which suppress IgE antibody production can be thought to be effective for the treatment of immediate type allergies. However, there are no effective drugs which suppress only IgE isotype antibody without suppressing other isotypes such as IgG, IgA so on,which are important for body defense mechanisms to various infectious diseases.
Accordingly, an object of the present invention is to provide an immunomodulator having suppressive activity on IgE production without any suppressive effect on other isotypes of antibody.
As a result of extensive investigation in order to solve the above-mentioned problem, the present investigators have unexpectedly found firstly that bacterial cells of Corynebacterium, Brevibacterium, and Microbacterium species, and mutant strains derived from the species are able to suppress selectively IgE type antibody production by oral administration of these bacterial cells without any suppressive effect on the production of other isotypes, even if enhancing these antibody production, and secondly that a decomposition product of above mentioned bacteria by enzymatic treatment and such, has more potent immunomodulative activity than that of the bacterial cells by themselves. These findings have led to the completion of the present invention.
That is, the present invention is to provide an immunomodulator, an immunomodulator food, immunomodulator drink containing bacterial cells belonging to Corynebacterium, Brevibacterium, and Microbacterium species, and mutant strains derived from the species, and a decomposition material of the above mentioned bacteria by enzymatic treatment and such.
The invention provides a method of modulating the immunological function by administering orally a pharmacologically effective amount of bacterial cells or their decomposition materials to a human being.
The invention provides a bacterial cell wall decomposition product (or decomposition material) having an immunomodulative activity, which is produced by dissolving the cell wall of at least one selected from the group consisting of bacteria belonging to Corynebacterium genera, Brevibacterium genera and Microbacterium genera and mutant strains of these bacteria.
The invention provides a process for manufacturing the above-shown bacterial cell wall decomposition product, which comprises dissolving the cell wall of at least one selected from the group consisting of bacteria belonging to Corynebacterium genera, Brevibacterium genera and Microbacterium genera and mutant strains of these bacteria.
It is preferable to the process to dissolve the cell walls with one of glucosidase and endopeptidase in the medium of an isotonic solution, separate the protoplast generated by the enzymatic digestion of the bacterial cells from the medium, and furthermore enzymatically treat the dissolved material of the bacterial cells with the other of the endopeptidase and glucosidase.
It is preferable to dissolve the cell walls with one of glucosidase and endopeptidase in the medium of an isotonic solution (biological saline), centrifuge the solution to obtain the supernatant and enzymatically treat the supernatant with the other of the endopeptidase and glucosidase.
The decomposition production of the invention may be obtained when at least 30% or 29% of the bacterial cells have been decomposed. This decomposition extent can be observed in terms of determination of dissolved hexosamine or glucosamine, which is present in the supernatant. Alternatively, it can be observed in terms of optical density of the bacterial suspension. The invention may reach at least 25% of optical density.
The invention provides an immunomodulator food or drink containing the bacterial cell wall decomposition product of the invention.
The invention provides a method of treating or preventing allergy, which comprises administering a pharmacologically effective amount of the decomposition product of the invention to a person suffering from allergy, and use of the decomposition product for manufacturing immunomodulator.
The invention relates to use of bacteria belonging to Corynebacterium genera, Brevibacterium genera and Microbacterium genera and mutant strains of these bacteria. That is, the invention provides a method of treating or preventing allergy, which comprises administering a pharmacologically effective amount of the bacteria to a person suffering from allergy; use of the bacteria for manufacturing immunomodulator; an Immunomodulator food or drink containing the bacteria; and a method of effecting immunomodulation, which comprises administering a pharmacologically effective amount of the bacteria to a person suffering from immunodeficiency.
It is preferred to the production of the decomposition product that the bacterial cell wall is digested with glucosidase and/or endopeptidase; a bacterium of Corynebacterium genus is Corynebacterium glutamicum or Corynebacterium herculis; a bacterium of Brevibacterium genus is Brevibacterium flavum; a bacterium of Microbacterium genus is Microbacterium ammoniaphilum; the glycosidase is egg white lysozyme or N-acetylmuramidase SG; the decomposition is performed by induction of lysogenic bacteriophage; the decomposition is performed by autolysis; the endopeptidase is bromelain; or the endopeptidase is one selected from the group consisting of seratiopeptidase, protease produced by Streptomyces griseus (K-1 strain), protease produced by Bacillus subtilis, Seaproze S produced by Armillaria mellea (Naratake in Japanese), trypsin, Achromobacter protease I and Grifola frondosa (Maitake in Japanese) metaloendopeptidase.
It is preferable that the immunomodulative bacterial cells are products produced by culturing cells of strains belonging to Corynebacterium, Brevibacterium, and Microbacterium species, and mutant strains derived from the species, heating the culture to kill the cells, harvesting the killed cells and dried. As for products of immunomodulative decomposition materials made of the above-mentioned bacteria, cultured cells are decomposed by autolysis, induction of lysogenic bacteriophage, and enzymatic treatment using cell wall digesting enzymes.
In the invention, decomposition material is defined by a generation of soluble hexosamine of at least 30% of the total bacterial hexosamine.
An immuno-adjuvant is called for as the substances which enhances antibody formation by injecting them mixed with an antigen to animals. Killed bacterial cells of Mycobacterium tuberculosis have been known to have immuno-adjuvant activity. Results of study for clarifying an effective component of the adjuvant activity revealed that the smallest component was muramyl-L-alanyl-D-isoglutamine (MDP), a component of peptidoglycan of bacterial cell wall. The existence of MDP is also known in all the bacterial cells other than bacteria of Mycobacterium tuberculosis, including pathogenic and also non-pathogenic bacteria, irrespective to classification by gram-positive and gram-negative staining. In fact, bacterial cell wall fractions containing MDP showed adjuvant activity in almost all bacteria studied (Kotani, S. Seikagaku 48, 1081-1107 1976 in Japanese).
A mucopeptide layer of bacterial cell wall is consisted of long glucosidase chains polymerized at xcex2-1,4 linkage of N-acetylglucosamine and muramic acid, and of peptide linkage which links calboxylic residue of muramic acid with alanine or L-glycine, D-glutamic acid, L-lysine or mesodiaminopimeritc acid, D-alanine in this turn. The last D-alanine makes peptide bond with a carboxic residue of another D-glutaminc acid, lysine and mesodiaminopimeric acid which is in the peptide chain originated from a neighboring glucoside chain. Number of amino acids in the peptide which links two neighboring glucoside chain are, somewhat different dependent on species of bacteria, 6 to 7 amino acids. Supposing that many glucoside chains arranged in parallel with each other are the warps, the peptide chains which bind the glucoside chains to each other are the wefts, therefore, both the wefts and the warps make a net-like structure and named peptidoglycan. The peptidoglycan surrounds cytoplasm of the bacterial cell and makes strong wall which protect the bacterial cell from damage by physical change of osmotic pressure.
The enzymes which solubilize the bacterial cell walls belong to the hydrolyze enzyme. Depending on their actin mechanisms, they are divided to three categories. One is glycosidase which hydrolyze carbohydrate linkage (for an instance egg-white lysozyme), others are endopeptidase which hydrolyze peptide bonds of peptidoglycan and amidase which hydrolyze a bond between muramic acid and amino acid.
Accordingly, MDP containing components are solubilized from bacterial cell wall by above-mentioned hydrolyze enzymes excluding amidase.
Being based on these background, various researches have been performed for the purpose of potentiation of immune status of the host. As the results, it has been reported studies on the resistance to tumor (Bogdanov I. G. et al. Antitumor glycopeptide from Lactobacillus bulgaricus cell wall FEBS LETT: 57(3)259-261 1975), activation of macrophage function and cellular immunity concerning anti-infectious immunity, and enhanced production of IgG antibody that plays important role for the resistance to bacterial and viral infections (Nammba Y et al. Effect of oral administration of lysozyme or digested cell wall on immunostimulation in guinea pigs. Infect Immun 31: 580-583 1981). As for relationships between chemical structure and biological activity, bacterial cell wall and its enzymatic digested material have been studied (Kotani, S. Seikagaku 48, 1081-1107 1976 in Japanese). However, no findings have been reported today as for production of IgE antibody which includes allergic disease such as atopy.
In the present invention, the bacteria are not necessarily special bacteria. However, in view of the safety and the utilization of waste material, amino acid-producing bacteria, for example, Corynebacterium glutamicum, Corynebacterium herculis, Corynebacterium fermentum, Brevibacterium flavum and Microbacterium ammoniaphilum can be mentioned.
The present inventors investigated effect of above-mentioned bacterial cells, a mutant strain of these bacteria, and enzymatically digested materials of these bacterial cells on IgE antibody production by oral administration, and have found that each bacterium suppressed IgE antibody production by administering heat-killed bacteria, enzymatically digested materials of the bacteria containing MDP, and that the IgE production suppressing activity of bacterial cells was potentiated by digesting the cells with cell-wall digesting enzymes.
Culture of bacteria belonging to genera of Corynebacterium, Brevibacterium and Microbacterium can be made by a known method in an appropriate medium. Any form of bacterial cell preparations can be used as a starting material such as the culture itself, centrifuged cells, freeze-dried cells, heat-killed cells and spray-dried cells for the purpose of production of the final raw products. Heat-killed cells or spray dried cells can be used as the IgE production immunomodulative material having suppressive effect on IgE production.
Production of the immunomodulative decomposition material starting from bacterial cell preparations is performed by solubilizing bacterial cell wall by means of using cell wall digesting enzymes, induction of lysogenic bacteriophage and autolysis using endogenous autolytic enzymes.
Of the methods mentioned above, an efficient method of decomposition of bacterial cell walls is the method using glycosidase and/or protease of endopeptidase type. The present invention revealed that the decomposition material produced by use of both of glycosidase and proteinase had more potent immunomodulative activity than that produced by use of either one of the two enzymes. Comparing the activity produced by two kinds of the enzymes, glycosidase is preferential. As an example of glycosidase, egg-white lysozyme, bacterial lysozyme and N-acetylmulamidase SG are mentioned. Of them, egg-white lysozyme is recommended from a stand point of safety of the product because this enzyme is used as a food additive.
As an example of the endopeptidase, bromelain, seratiopeptidase, protease produced by Streptomyces griseus K-1 (Pronase(copyright)), protease produced by Bacillus subtilis (Nagase(copyright)), Seaprose S produced by Armillaria mellea (Naratake in Japanese), Acromobacter protease 1 (lysyl-endopeptidase) and metalloendopeptidase produced by Grifola frondosa (Maitake in Japanese) can be mentioned. Of these, bromelain is recommended from a stand point of safety of the product because this enzyme is used as a food additive.
Sensitivity of bacterial cells to glycosidase digestion differs depending on the bacterial strain. The sensitivity can be enhanced by addition of penicillin or glycine in culture medium as described in Examples 1 and 2. Enzyme treatment is conducted under nearly optimal pH of the respective enzymes. The treatment by glycosidase and endopeptidase is performed under nearly neutral pH, i.e., pH 5 to 8. An amount of enzyme added to 1 gm of dry bacterial cells i s 0.01 to 10 mg. Temperature of enzyme treatment can be conducted at room temperature to 70xc2x0 C. DNase and RNase at concentrations of 10 to 500 ug/ml can be added to the reaction mixture to reduce viscosity of the mixture.
Decomposition of bacterial cell by an induction of lysogenic bacteriophage can be done by UV irradiation of each of bacterial culture of Corynebacterium, Brevibacterium and Microbacterium. Decomposition of bacterial cell by autolysis can be performed by incubation of bacterial cell suspended in purified water at about 50xc2x0 C. for 1 to 3 days.
A definite method for production of the immunomodulative decomposed material of the present invention is, for example, the following. Corynebacterium glutamicum is cultured in appropriate medium. The culture is centrifuged to collect bacterial cells. The cells are suspended in physiological isotonic solution such as physiological saline, and then either of glycosidase such as egg-white lysozyme or endopeptidase such as bromelain is added to the suspension to solubilize the bacterial cell wall. Bacterial cytoplasmic membrane and cytoplasm surrounded by the cytoplasmic membrane exist as protoplasts by this stage of enzyme treatment. The protoplast is sedimented by centrifugation and discarded. Then, the supernatant, which contains MDP containing cell wall components, is treated with another enzyme, i.e., endopeptidase (if enzyme used at the first step is glycosidase) such as bromeline or glycosidase (if enzyme used at the first step is endopeptidase) such as egg white lysozyme.
A preferable method is the following. At first, the bacterial cell wall is solubilized by treatment with glycosidase and protoplast is sedimented and discarded and then endopeptidase treatment of the supernatant solution is conducted. Elimination of the protoplast effectively purify the active decomposed material of the present invention as in the Examples. The immunomodulative active decomposition material can be precipitated by adding ethanol or acetone to the supernatant.
Other method of preparation of the present invention is the following. Corynebacterium glutamicum is cultured and the bacterial cells are harvested and suspended in purified water. Then, at first, glycosidase is added to the suspension and incubated to lyse cell wall and then endopeptidase is added to the suspension and incubated.
The process of,decomposition of bacterial cells is measured by the estimation of optical density of bacterial suspension at wavelengths of 600 to 660 nm as described in Example 7 or by hexosamine determination of supernatant solution after centrifugation of bacterial suspension as described in Example 4. Hexosamine determination can be conducted by ordinary methods such as the Elson-Morgan Method and the Reissig, Strominger and Leloir Method.
An enhanced activity on immunomodulation owing to decomposition of bacterial cells is observed when at least 30% of bacterial cells have been decomposed in terms of dissolved hexosamine determination. Alternatively, at least 25% reduction of optical density of the bacterial suspension is preferably found for this purpose as in Example 4 .
A standard oral daily dose of the immunomodulative bacterial cells and their decomposition material of the present invention is between 0.2 and 2 g. The desirable dose is about 1 g equivalent to killed dry cells.
When the immunomodulative decomposition material of the present invention is applied as a main agent, it can orally be used in powder, tablets, dispersion, capsules, confectionery, bread, noodles, drinks or the like. These products can be made by ordinary production methods.